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1.
《Biophysical journal》2021,120(24):5644-5656
Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. Although most of these disease-linked variants cause loss of function because of misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Coexpression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Furthermore, when T39R was coexpressed with Cx46, it produced hemichannels with increased activity, particularly at negative potentials, which could potentially contribute to its pathogenicity in the lens. In contrast, coexpression of wild-type Cx50 with Cx46 was associated with a marked reduction in hemichannel activity, indicating that it may have a protective effect. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal (NT) domain while also neutralizing the voltage-sensing residue D3 as well as residue E42, which participates in loop gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts. 相似文献
2.
Hoa T. Le Wun Chey Sin Shannon Lozinsky John Bechberger José Luis Vega Xu Qiu Guo Juan C. Sáez Christian C. Naus 《The Journal of biological chemistry》2014,289(3):1345-1354
Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states. 相似文献
3.
Intracellular distribution, assembly and effect of disease-associated connexin 31 mutants in HeLa cells 总被引:3,自引:0,他引:3
He LQ Liu Y Cai F Tan ZP Pan Q Liang DS Long ZG Wu LQ Huang LQ Dai HP Xia K Xia JH Zhang ZH 《Acta biochimica et biophysica Sinica》2005,37(8):547-554
Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV),hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD,141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I 141V and 652del 12). Immunoblotting analysis showed that 12 mutated Cx31 s, like WTCx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) caused cell death in HeLa cells. However, the five heating impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, diseaseassociated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants. 相似文献
4.
Mese G Sellitto C Li L Wang HZ Valiunas V Richard G Brink PR White TW 《Molecular biology of the cell》2011,22(24):4776-4786
Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders. 相似文献
5.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2020,1865(8):158705
Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity. 相似文献
6.
It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels. 相似文献
7.
《Cell communication & adhesion》2013,20(4-6):245-249
It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was ~220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of ~75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels. 相似文献
8.
Connexins are mechanosensitive 总被引:11,自引:0,他引:11
Connexins form gap junction channels that provide a hydrophilic path between cell interiors. Some connexins, particularly the lens connexins, Cx46 and Cx50 and their orthologs, can form functional hemichannels in nonjunctional membranes. These hemichannels are a nonselective conduit to the extracellular medium and may jeopardize cell survival. The physiological function of hemichannels has remained elusive, but it has been postulated that hemichannels are involved in ATP-release caused by mechanical stimulation. Here we show with single-channel and whole cell electrophysiological studies that Cx46 hemichannels are mechanosensitive, like other families of ion channels and membrane-bound enzymes. The hemichannel response to mechanical stress is bipolar. At negative potentials stress opens the channel, and at positive potentials stress closes it. Physiologically, Cx46 hemichannels may assist accommodation of the ocular lens by providing a transient path for volume flow as the lens changes shape. mechanical stress; lens 相似文献
9.
Nidhi Batra Manuel A. Riquelme Sirisha Burra Rekha Kar Sumin Gu Jean X. Jiang 《The Journal of biological chemistry》2014,289(15):10582-10591
Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone. 相似文献
10.
Srinivas M Calderon DP Kronengold J Verselis VK 《The Journal of general physiology》2006,127(1):67-75
Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+. 相似文献
11.
Srinivas M Kronengold J Bukauskas FF Bargiello TA Verselis VK 《Biophysical journal》2005,88(3):1725-1739
Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties. 相似文献
12.
Although a functional pore domain is required for connexin 37 (Cx37)–mediated suppression of rat insulinoma (Rin) cell proliferation, it is unknown whether functional hemichannels would be sufficient or if Cx37 gap junction channels are required for growth suppression. To test this possibility, we targeted extracellular loop cysteines for mutation, expecting that the mutated protein would retain hemichannel, but not gap junction channel, functionality. Cysteines at positions 61 and 65 in the first extracellular loop of Cx37 were mutated to alanine and the mutant protein (Cx37-C61,65A) expressed in Rin cells. Although the resulting iRin37-C61,65A cells expressed the mutant protein comparably to Cx37 wild-type (Cx37-WT)–expressing Rin cells (iRin37), Cx37-C61,65A expression did not suppress the proliferation of Rin cells. As expected, iRin37-C61,65A cells did not form functional gap junction channels. However, functional hemichannels also could not be detected in iRin37-C61,65A cells by either dye uptake or electrophysiological approaches. Thus, failure of Cx37-C61,65A to suppress the proliferation of Rin cells is consistent with previous data demonstrating the importance of channel functionality to Cx37’s growth-suppressive function. Moreover, failure of the Cx37-C61,65A hemichannel to function, even in low external calcium, emphasizes the importance of extracellular loop cysteines not only in hemichannel docking but also in determining the ability of the hemichannel to adopt a closed configuration that can open in response to triggers, such as low external calcium, effective at opening Cx37-WT hemichannels. 相似文献
13.
Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin 总被引:5,自引:0,他引:5
Cherian PP Siller-Jackson AJ Gu S Wang X Bonewald LF Sprague E Jiang JX 《Molecular biology of the cell》2005,16(7):3100-3106
Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain. 相似文献
14.
Nakagawa S Gong XQ Maeda S Dong Y Misumi Y Tsukihara T Bai D 《The Journal of biological chemistry》2011,286(22):19672-19681
The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels. 相似文献
15.
Lens fiber connexins, cx50 and cx46 (alpha3 and alpha8), belong to a small subset of connexins that can form functional hemichannels in nonjunctional membranes. Knockout of either cx50 or cx46 results in a cataract, so the properties of both connexins are likely essential for proper physiological functioning of the lens. Although portions of the sequences of these two connexins are nearly identical, their hemichannel properties are quite different. Cx50 hemichannels are much more sensitive to extracellular acidification than cx46 hemichannels and differ from cx46 hemichannels both in steady-state and kinetic properties. Comparison of the two branches of the cx50 hemichannel G-V curve with the junctional G-V curve suggests that cx50 gap junctions gate with positive relative polarity. The histidine-modifying reagent, diethyl pyrocarbonate, reversibly blocks cx50 hemichannel currents but not cx46 hemichannel currents. Because cx46 and cx50 have very similar amino acid sequences, one might expect that replacing the two histidines unique to the third transmembrane region of cx50 with the corresponding cx46 residues would produce mutants more closely resembling cx46. In fact this does not happen. Instead the mutant cx50H161N does not form detectable hemichannels but forms gap junctions indistinguishable from wild type. Cx50H176Q is oocyte lethal, and the double mutant, cx50H61N/H176Q, neither forms hemichannels nor kills oocytes. 相似文献
16.
Extracellular-loop peptide antibodies reveal a predominant hemichannel organization of connexins in polarized intestinal cells 总被引:3,自引:0,他引:3
Clair C Combettes L Pierre F Sansonetti P Tran Van Nhieu G 《Experimental cell research》2008,314(6):1250-1265
Shigella, the causative agent of bacillary dysentery, invades colonic epithelial cells to elicit an intense inflammatory reaction leading to destruction of the mucosa. ATP-dependent paracrine signalling induced by connexin (Cx) hemichannel opening was previously shown to favor Shigella flexneri invasion and dissemination in transfectants of HeLa cells [G. Tran Van Nhieu, C. Clair, R. Bruzzone, M. Mesnil, P. Sansonetti and L. Combettes. (2003). Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells. Nat. Cell Biol. 5, 720-726.]. However, although Cxs have been described in polarized epithelial cells, little is known about their structural organization and the role of hemichannels during S. flexneri invasion. We show here that polarized Caco-2/TC7 cells express significant amounts of Cx26, Cx32 and Cx43, but that unexpectedly, cell-cell coupling assessed by dye-transfer experiments is inefficient. Consistent with a predominant Cx organization in hemichannels, dye loading induced by low calcium was readily observed, with preferential loading at the basolateral side. Antibodies (Abs) against connexin extracellular loop peptides (CELAbs) demonstrated the importance of hemichannel signalling since they inhibited dye uptake at low calcium and at physiological calcium concentrations during S. flexneri invasion. Importantly, CELAbs allowed the visualization of hemichannels at the surface of epithelial cells, as structures distinct from gap intercellular junctions. 相似文献
17.
Oculodentodigital dysplasia (ODDD) is a dominantly inherited human disorder associated with different symptoms like craniofacial
anomalies, syndactyly and heart dysfunction. ODDD is caused by mutations in the GJA1 gene encoding the gap junction protein connexin43 (Cx43). Here, we have characterized four Cx43 mutations (I31M, G138R, G143S
and H194P) after stable expression in HeLa cells. In patients, the I31M and G138R mutations showed all phenotypic characteristics
of ODDD, whereas G143S did not result in facial abnormalities and H194P mutated patients exhibited no syndactylies. In transfected
HeLa cells, these mutations led to lack of the P2 phosphorylation state of the Cx43 protein, complete inhibition of gap junctional
coupling measured by neurobiotin transfer and increased hemichannel activity. In addition, altered trafficking and delayed
degradation were found in these mutants by immunofluorescence and pulse-chase analyses. In G138R and G143S mutants, the increased
hemichannel activity correlated with an increased half-time of the Cx43 protein. However, the I31M mutated protein showed
no extended half-time. Thus, the increased hemichannel activity may be directly caused by an altered conformation of the mutated
channel forming protein. We hypothesize that increased hemichannel activity may aggravate the phenotypic abnormalities in
ODDD patients who are deficient in Cx43 gap junction channels.
Radoslaw Dobrowolski and Annette Sommershof contributed equally to this work. 相似文献
18.
R Ponsaerts C D'hondt F Hertens JB Parys L Leybaert J Vereecke B Himpens G Bultynck 《PloS one》2012,7(7):e42074
ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress. 相似文献
19.
Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions. 相似文献
20.
Structural determinants for the differences in voltage gating of chicken Cx56 and Cx45.6 gap-junctional hemichannels
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The voltage- and calcium-dependent gating properties of two lens gap-junctional hemichannels were compared at the macroscopic and single channel level. In solutions containing zero added calcium and 1 mM Mg, chicken Cx56 hemichannels were mostly closed at negative potentials and application of depolarizing voltage clamp steps elicited a slowly activating outward current. In contrast, chicken Cx45.6 hemichannels were predominantly open at negative potentials and rapidly closed in response to application of large depolarizing potentials. Another difference was that macroscopic Cx45.6 currents were much smaller in size than the hemichannel currents induced by oocytes with similar amounts of cRNA for Cx56. The aim of this study was to identify which regions of the connexins were responsible for the differences in voltage-dependent gating and macroscopic current amplitude by constructing a series of chimeric Cx45.6-Cx56 channels. Our results show that two charged amino acids that are specific for the alpha3-group connexins (R9 in the N-terminus and E43 in the first extracellular loop) are important determinants for the difference in voltage-dependent gating between Cx45.6 and Cx56 hemichannels; the first transmembrane-spanning domain, M1, is an important determinant of macroscopic current magnitude; R9 and E43 are also determinants of single channel conductance and rectification. 相似文献